Process for Preparing Imetelstat

ABSTRACT

The present invention relates to a process for preparing the telomerase inhibitor imetelstat using a 3 steps per cycle solid-phase support bound process comprising the steps of deprotection of the 3′-amino group of the support-bound oligonucleotide, coupling with a 5′-phosphoramidite, and sulfurization with an acyl disulfide, characterized by the absence of an additional capping step in each cycle that is used to prevent unreacted 3′-amino oligonucleotide groups from reacting during subsequent cycles. Imetelstat has formula below.

The present invention relates to a process for preparing the telomeraseinhibitor imetelstat using a 3 steps per cycle solid-phase support boundprocess comprising the steps of deprotection of the 3′-amino group ofthe support-bound oligonucleotide, coupling with a 5′-phosphoramidite,and sulfurization with an acyl disulfide, characterized by the absenceof an additional capping step in each cycle that is used to preventunreacted 3′-amino oligonucleotide groups from reacting duringsubsequent cycles.

BACKGROUND

Imetelstat (SEQ ID NO:1) is a N3′→P5′ thiophosphoramidateoligonucleotide covalently linked to a palmitoyl lipid moiety and hasbeen described in WO-2005/023994 as compound (1F). The sodium salt ofimetelstat acts as a potent and specific telomerase inhibitor and can beused to treat telomerase-mediated disorders, e.g. cancer, includingdisorders such as myelofibrosis (MF), myelodysplastic syndromes (MDS)and acute myelogenous leukemia (AML).

The structure of imetelstat sodium is shown below:

The structure of imetelstat can also be represented as shown below

imetelstat

B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G B₆ =T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C = cytosine

The LPT group represents the palmitoyl lipid that is covalently linkedto the N3′→P5′ thiophosphor-amidate oligonucleotide. The base sequenceof the thirteen nucleotides is as follows: TAGGGTTAGACAA and isrepresented by the bases B₁ to B₁₃. The —NH—P(═S)(OH)— and —O—P(═S)(OH)—groups of the structure can occur in a salt form. It is understood thatsalt forms of a subject compound are encompassed by the structuresdepicted herein, even if not specifically indicated.

Imetelstat sodium can also be represented as follows:

  B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G  B₆ = T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C =cytosine

The —NH—P(═S)(OH)— group and the thymine, adenine, guanine and cytosinebases can occur in other tautomeric arrangements then used in thefigures of the description. It is understood that all tautomeric formsof a subject compound are encompassed by a structure where one possibletautomeric form of the compound is described, even if not specificallyindicated.

PRIOR ART

The synthetic scheme used in WO-2005/023994 to prepare imetelstat ascompound (1F) is described in Scheme 1 and Scheme 2. The synthesis ofthis oligonucleotide is achieved using the solid-phase phosphoramiditemethodology with all reactions taking place on solid-phase support. Thesynthesis of imetelstat is carried out on controlled pore glass(LCAA-CPG) loaded with3-palmitoylamido-1-O-(4,4′-dimethoxytrityl)-2-O-succinyl propanediol.The oligonucleotide is assembled from the 5′ to the 3′ terminus by theaddition of protected nucleoside 5′-phosphor-amidites with theassistance of an activator. Each elongation cycle consists of 4distinct, highly controlled steps: deprotection, amidite coupling,sulfurization and a capping step.

In Scheme 1 the solid-phase supported synthesis starts with removal ofthe acid-labile 4,4-dimethoxy-trityl (DMT) protecting group from thepalmitoylamidopropanediol linked to the solid-phase support. The firstphosphoramidite nucleotide is coupled to the support followed bysulfurization of the phosphor using a 0.1 M solution of phenylacetyldisulfide (PADS) in a mixture of acetonitrile and 2,6-lutidine (1:1ratio). Then a capping step is applied to prevent any unreactedsolid-phase support starting material from coupling with aphosphoramidite nucleotide in the following reaction cycles. Capping isdone using an 18:1:1 mixture of THF/isobutyric anhydride/2,6-lutidine.

After the first cycle on the solid-phase support, chain elongation isachieved by reaction of the 3′-amino group of the support-boundoligonucleotide with an excess of a solution of the protected nucleotidephosphoramidite monomer corresponding to the next required nucleotide inthe sequence as depicted in Scheme 2.

In Scheme 2 the first cycle is depicted of the chain elongation processwhich is achieved by deprotection of the 3′-amino group of thesupport-bound oligonucleotide (a), followed by a coupling reaction ofthe 3′-amino group of the support-bound oligonucleotide (b) with anexcess of a solution of a 5′-phosphoramidite monomer corresponding tothe next required nucleotide in the sequence of imetelstat. The couplingreaction is followed by sulfurization of the phosphor of thesupport-bound oligonucleotide (c) and a capping step (see Scheme 3) toprevent any unreacted solid-phase support starting material (b) fromcoupling with a 5′-phosphoramidite nucleotide in the following reactioncycles. The reaction cycle of Scheme 2 is repeated 12 times before thesolid-phase support-bound oligonucleotide is treated with a 1:1 mixtureof ethanol and concentrated ammonia, followed by HPLC purification toobtain imetelstat.

The capping step using an 18:1:1 mixture of THF/isobutyricanhydride/2,6-lutidine is done to convert after the coupling step anyremaining solid-phase support bound oligonucleotide (b) with a primary3′-amino group into oligonucleotide (e) with a protected (or ‘capped’)3′-amino group in order to prevent the primary 3′-amino group fromcoupling with a phosphoramidite nucleotide in the next reaction cycles.

WO-01/18015 discloses in Example 3 with SEQ ID No. 2 a N3′→P5′thiophosphoramidate oligonucleotide and a process for preparing thisoligonucleotide encompassing a capping step.

Herbert B-S et al. discusses the lipid modification of GRN163 (Oncogene(2005) 24, 5262-5268).

Makiko Horie et al. discusses the synthesis and properties of2′-O,4′-C-ethylene-bridged nucleic acid oligonucleotides targeted tohuman telomerase RNA subunit (Nucleic Acids Symposium Series (2005) 49,171-172).

DESCRIPTION OF THF INVENTION

The coupling reaction in the solid-phase support bound process disclosedin WO-01/18015 and WO-2005/023994 include a capping step to prevent anyunreacted primary 3′ amino groups on the support-bound oligonucleotidefrom reacting during subsequent cycles.

It has now surprisingly been found that the use of a capping step asdescribed in the prior art is superfluous and that imetelstat can beprepared using a 3-step cycle without an additional capping step withnearly identical yield and purity compared to the prior art 4-step cyclethat uses a specific capping step. Eliminating the capping step fromeach cycle benefits the overall process by reducing the number of cyclesteps by 22% (from 54 to 42 steps) and consequent reduction of processtime. Also, the solvent consumption is reduced due to the reduction ofcycle steps which makes for a greener process.

Wherever the term “capping step” is used throughout this text, it isintended to define an additional chemical process step wherein theprimary free 3′-amino group on the solid-phase support boundoligonucleotide is converted into a substituted secondary or tertiary3′-amino group that is not capable of participating in the couplingreaction with a protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramiditemonomer in the ensuing coupling step.

In one embodiment, the present invention relates to a method ofsynthesizing an oligonucleotide N3′→P5′ thiophosphoramidate of formula

imetelstat

  B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G  B₆ = T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C =cytosinethe method comprises of

-   a) providing a first 3′-amino protected nucleotide attached to a    solid-phase support of formula (A) wherein PG is an acid-labile    protecting group;

-   b) deprotecting the protected 3′-amino group to form a free 3′-amino    group;

-   c) reacting the free 3′-amino group with a protected    3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite    monomer of formula (B′_(n)) wherein n=2 to form an internucleoside    N3′→P5′-phosphoramidite linkage;

-   d) sulfurization of the internucleoside phosphoramidite group using    an acyl disulfide to form a N3′→P5′ thiophosphoramidate;-   e) repeating 11 times in successive order the deprotection step b),    the coupling step c) with a protected    3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramidite    monomer of formula (B′_(n)) wherein the protected nucleoside base B′    in monomer (B′_(n)) is successively the protected nucleobase B₃ to    B₁₃ in the respective 11 coupling steps, and the sulfurization step    d);-   f) removing the acid-labile protecting group PG; and-   g) cleaving and deprotecting imetelstat from the solid-phase    support;    characterized in that no additional capping step is performed in any    of the reaction steps a) to e).-   In one embodiment, the present invention relates to a method of    synthesizing the N3′→P5′ thiophosphoramidate oligonucleotide    imetelstat of formula

imetelstat

  B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G  B₆ = T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C =cytosine

-   -   the method comprises of    -   a) providing a first 3′-amino protected nucleotide attached to a        solid-phase support of formula (A) wherein PG is an acid-labile        protecting group;

-   -   b) deprotecting the protected 3′-amino group to form a free        3′-amino group;

-   -   c) reacting the free 3′-amino group with a protected        3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite        monomer of formula (B′_(n)), wherein B′_(n) with n=2 is        protected A, to form an internucleoside N3′→P5′-phosphoramidite        linkage;

-   -   d) sulfurization of the internucleoside phosphoramidite group        using an acyl disulfide to form a N3′→P5′ thiophosphoramidate;    -   e) repeating 11 times in successive order the deprotection step        b), the coupling step c) with a protected        3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramidite        monomer of formula (B′_(n)) wherein the nucleoside base B′ of        monomer (B′_(n)) is protected B except when B is thymine, and        wherein B_(n) is successively nucleobase B₃ to B₁₃ in the        respective 11 coupling steps, and the sulfurization step d);    -   f) removing the acid-labile protecting group PG; and    -   g) deprotecting and cleaving imetelstat from the solid-phase        support;    -   characterized in that no additional capping step is performed in        any of the reaction steps a) to e).

-   In one embodiment, the present invention relates to a method of    synthesizing the N3′→P5′ thiophosphoramidate oligonucleotide    imetelstat of formula

imetelstat

  B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G  B₆ = T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C =cytosine

-   -   the method comprises of    -   a) providing a first protected 3′-amino nucleotide attached to a        solid-phase support of formula (A) wherein PG is an acid-labile        protecting group;

-   -   b) deprotecting the PG-protected 3′-amino nucleotide to form a        free 3′-amino nucleotide of formula (A′);

-   -   c) coupling the free 3′-amino nucleotide with a protected        3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite        monomer (B′_(n)), wherein B′_(n) with n=2 is protected A, to        form an internucleoside N3′→P5′-phosphoramidite linkage;

-   -   d) sulfurizing the N3′→P5′-phosphoramidite linkage using an acyl        disulfide to form an internucleoside N3′→P5′ thiophosphoramidate        linkage;    -   e) repeating 11 times in successive order:        -   the deprotecting step b);        -   the coupling step c) with a protected            3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramidite            monomer (B′_(n)) wherein the nucleoside base B′ of monomer            (B′_(n)) is protected B except when B is thymine, and            wherein B_(n) is successively nucleobase B₃ to B₁₃ in the            respective 11 coupling steps; and the sulfurizing step d);        -   to produce a protected N3′→P5′ thiophosphoramidate            oligonucleotide imetelstat attached to the solid-phase            support;    -   f) removing the 3′-terminal acid-labile protecting group PG from        the protected N3′→P5′ thiophosphoramidate oligonucleotide        imetelstat; and    -   g) deprotecting and cleaving the protected N3′→P5′        thiophosphoramidate oligonucleotide imetelstat from the        solid-phase support to produce imetelstat;    -   characterized in that no additional capping step is performed in        any of the reaction steps a) to e).

A wide variety of solid-phase supports may be used with the invention,including but not limited to, such as microparticles made of controlledpore glass (CPG), highly cross-linked polystyrene, hybrid controlledpore glass loaded with cross-linked polystyrene supports, acryliccopolymers, cellulose, nylon, dextran, latex, polyacrolein, and thelike.

The 3′-amino protected nucleotide attached to a solid-phase support offormula (A)

can be prepared as disclosed in WO-2005/023994 wherein a controlled poreglass support loaded with3-palmitoylamido-1-O-(4,4′-dimethoxytrityl)-2-O-succinyl propanediol hasbeen coupled with a protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramiditemonomer of formula (B′₁)

wherein PG is an acid-labile protecting group. Suitable acid-labile3′-amino protecting groups PG are, but not limited to, e.g.triphenylmethyl (i.e. trityl or Tr), p-anisyldiphenylmethyl (i.e.mono-methoxytrityl or MMT), and di-p-anisylphenylmethyl (i.e.dimethoxytrityl or DMT).

The protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramiditemonomers of formula (B′_(n)) have a 3′-amino protecting group PG whichis an acid-labile group, such as triphenylmethyl (i.e. trityl or Tr),p-anisyldiphenylmethyl (i.e. monomethoxytrityl or MMT), ordi-p-anisylphenylmethyl (i.e. dimethoxytrityl or DMT). Furthermore thenucleoside base B′ is protected with a base-labile protecting group(except for thymine).

  B′₁ = T B′₁₀ = protected A B′₂ = protected A B′₁₁ = protected C B′₃ =protected G B′₁₂ = protected A B′₄ = protected G B′₁₃ = protected A B′₅= protected G   B′₆ = T T = thymine B′₇ = T A = adenine B′₈ = protectedA G = guanine B′₉ = protected G C = cytosine

The nucleotide monomers B′₁ and B′₂ to B′₁₃ are used successively in the13 coupling steps starting from the provision of a solid-phase supportloaded with 3-palmitoylamido-1-O-(4,4′-dimethoxytrityl)-2-O-succinylpropanediol and coupled to nucleotide monomer B′₁ and the followingcycle of 12 deprotection, coupling, and sulfurization reactions whereinthe nucleotide monomers B′₂ to B′₁₃ are used.

The 3′-amino protecting group PG can be removed by treatment with anacidic solution such as e.g. dichloroacetic acid in dichloromethane ortoluene.

The nucleoside base B′ in the protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropyl-aminophosphoramiditemonomers of formula (B′_(n)) is protected with a base-labile protectinggroup which is removed in step g). Suitable base-labile protectinggroups for the nucleoside base adenine, cytosine or guanine are e.g.acyl groups such as acetyl, benzoyl, isobutyryl, dimethyl-formamidinyl,or dibenzylformamidinyl. Under the reaction conditions used inoligonucleotide synthesis the thymine nucleoside base does not requireprotection. Such protected3′-amino-nucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramiditemonomers of formula (B′_(n)) having a 3′-amino protected with anacid-labile group protecting group PG and a nucleoside base B′ protectedwith a base-labile protecting group are commercially available or can beprepared as described in WO-2006/014387.

The coupling step c) is performed by adding a solution of protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramiditemonomer of formula (B_(n)) and a solution of an activator (or a solutioncontaining the phosphoramidite monomer (B_(n)) and the activator) to thereaction vessel containing the free amino group of an (oligo)nucleotidecovalently attached to a solid support. The mixture is then mixed bysuch methods as mechanically vortexing, sparging with an inert gas, etc.Alternately, the solution(s) of monomer and activator can be made toflow through a reaction vessel (or column) containing the solid-phasesupported (oligo)nucleotide with a free 3′-amino group. The monomer andthe activator either can be premixed, mixed in the valve-block of asuitable synthesizer, mixed in a pre-activation vessel andpreequilibrated if desired, or they can be added separately to thereaction vessel.

Examples of activators for use in the invention are, but not limited to,tetrazole, 5-(ethylthio)-1H-tetrazole, 5-(4-nitro-phenyl)tetrazole,5-(2-thienyl)-1H-tetrazole, triazole, pyridinium chloride, and the like.Suitable solvents are acetonitrile, tetrahydrofuran, dichloromethane,and the like. In practice acetonitrile is a commonly used solvent foroligonucleotide synthesis.

The sulfurization agent for use in step d) is an acyl disulfidedissolved in a solvent. Art know acyl disulfides are e.g. dibenzoyldisulphide, bis(phenylacetyl) disulfide (PADS), bis(4-methoxybenzoyl)disulphide, bis(4-methylbenzoyl) disulphide, bis(4-nitrobenzoyl)disulphide and bis(4-chlorobenzoyl) disulfide.

Phenylacetyl disulfide (PADS) is a commonly used agent for sulfurizationreactions that it is best ‘aged’ in a basic solution to obtain optimalsulfurization activity (Scotson J. L. et al., Org. Biomol. Chem., vol.14, 10840-10847, 2016). A suitable solvent for PADS is e.g. a mixture ofa basic solvent such as e.g. 3-picoline or 2,6-lutidine with aco-solvent such as acetonitrile, toluene, 1-methyl-pyrrolidinone ortetrahydrofuran. The amount of the basic solvent to the amount of theco-solvent can be any ratio including a 1:1 ratio. Depending upon thephosphite ester to be converted into its corresponding thiophospate,both ‘fresh’ and ‘aged’ PADS can be used however ‘aged’ PADS has beenshown to improve the rate and efficiency of sulfurization. ‘Aged’ PADSsolutions are freshly prepared PADS solutions that were maintained sometime before usage in the sulfurization reaction. Aging times can varyfrom a few hours to 48 hours and the skilled person can determine theoptimal aging time by analysing the sulfurization reaction for yield andpurity.

For the preparation of imetelstat in accordance with the presentinvention, a PADS solution in a mixture of acetonitrile and2,6-lutidine, preferably in a 1:1 ratio, with an aging time of 4 to 14hours is used. It has been found that when 2,6-lutidine is used,limiting the amount of 2,3,5-collidine (which is often found as animpurity in 2,6-lutidine) below 0.1% improves the efficiency ofsulfurization and less undesirable phosphor oxidation is observed.

In step g) imetelstat is deprotected and cleaved from the solid-phasesupport. Deprotection includes the removal of the β-cyanoethyl groupsand the base-labile protecting groups on the nucleotide bases. This canbe done by treatment with a basic solution such as a diethylamine (DEA)solution in acetonitrile, followed by treatment with aqueous ammoniadissolved in an alcohol such as ethanol.

The reaction steps a) to f) of the present invention are carried out inthe temperature range of 10° C. to 40° C. More preferably, thesereactions are carried out at a controlled temperature ranging from 15°C. to 30° C. In particular reaction step b) of the present invention iscarried out in the temperature range of 15° C. to 30° C.; more inparticular 17° C. to 27° C. In particular reaction step d) of thepresent invention is carried out in the temperature range of 17° C. to25° C.; more in particular 18° C. to 22° C.; even more in particular 19°C. The step g) wherein imetelstat is deprotected and cleaved from thesolid-phase support is carried out at a temperature ranging from 30° C.to 60° C. Depending upon the equipment and the specific reactionconditions used, the optimal reaction temperature for each step a) to g)within the above stated ranges can be determined by the skilled person.

After each step in the elongation cycle, the solid-phase support isrinsed with a solvent, for instance acetonitrile, in preparation for thenext reaction.

After step g), crude imetelstat is obtained in its ammonium salt formwhich is then purified by a preparative reversed phase high performanceliquid chromatography (RP-HPLC) by using either polymeric or silicabased resins to get purified imetelstat in triethyl amine form. Anexcess of a sodium salt is added, and then the solution is desalted bydiafiltration thereby yielding imetelstat sodium which is thenlyophilized to remove water.

EXPERIMENTAL PART

‘Room temperature’ or ‘ambient temperature’ typically is between 21-25°C.

Experiment 1 (No Capping Step)

All the reagents and starting material solutions were prepared including3% dichloroacetic acid (DCA) in toluene, 0.5 M5-(ethylthio)-1H-tetrazole in acetonitrile, 0.15 M of all 4 nucleotidemonomers of formula (B′_(n)) in acetonitrile, 0.2 M phenyl acetyldisulfide (PADS) in a 1:1 mixture of acetonitrile and 2,6-lutidine and20% DEA (diethylamine) in acetonitrile.

nucleotide monomer of formula (B’_(n)) Structure B’₁, B’₆, B’₇

B’₂, B’₈, B’₁₀, B’₁₂, B’₁₃

B’₃, B’₄, B’₅, B’₉

B’₁₁

The oligonucleotide synthesis was performed in the direction of 5′ to 3′utilizing a repetitive synthesis cycle consisting of detritylationfollowed by coupling, and sulfurization performed at ambienttemperature.

A column (diameter: 3.5 cm) was packed with a solid-support loaded with3-palmitoylamido-1-O-(4,4′-dimethoxytrityl)-2-O-succinyl propanediol(3.5 mmol based on a capacity of 400 μmol/g) that was coupled with thenucleotide monomer B′₁. Detritylation was achieved using 3%dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4column volumes in each detritylation step) and the solid-support boundnucleotide was washed with acetonitrile (amount: 5 column volumes).Coupling with the next nucleotide monomer of formula (B′_(n))) wasachieved by pumping a solution of 0.5 M 5-(ethylthio)-1H-tetrazole inacetonitrile and 0.15 M of the next nucleotide monomer of formula(B′_(n)) in the sequence, dissolved in acetonitrile, through the column.The column was washed with acetonitrile (amount: 2 column volumes). Thensulfurization was performed by pumping a solution of 0.2 M phenyl acetyldisulfide (PADS) in a 1:1 mixture of acetonitrile and 2,6-lutidinemixture through the column followed by washing the column withacetonitrile (amount: 5 column volumes).

The synthesis cycle of detritylation, coupling with the next nucleotidemonomer of formula (B′_(n)) and sulfurization was repeated 12 times,followed by detritylation using 3% dichloroacetic acid (DCA) in toluene(amount is between 6.5 and 13.4 column volumes).

Upon completion of the synthesis cycle, the crude oligonucleotide on thesolid-support support was treated with a diethylamine (DEA) solutionfollowed by treatment with ammonium hydroxide solution: ethanol (3:1volume ratio) at a temperature of 55° C. The reaction mixture was agedfor 4 to 24 hours at 55° C., cooled to room temperature, and slurry wasfiltered to remove the polymeric support. The solution comprisingimetelstat in its ammonium form was subjected to the HPLC analysisprocedure of Experiment 3.

Experiment 2 (with Capping Step)

All the reagents and starting material solutions were prepared including3% dichloroacetic acid (DCA) in toluene, 0.5 M5-(ethylthio)-1H-tetrazole in acetonitrile, 0.15 M of all 4 nucleotidemonomers of formula (B′_(n)) in acetonitrile, 0.2 M phenyl acetyldisulfide (PADS) in a 1:1 mixture of acetonitrile and 2,6-lutidinemixture, 20% N-methylimidazole (NMI) in acetonitrile as capping agent A,isobutryic anhydride in a 1:1 mixture of acetonitrile and 2,6-lutidinemixture as capping agent B and 20% DEA in acetonitrile.

The oligonucleotide synthesis was performed in the direction of 5′ to 3′utilizing a repetitive synthesis cycle consisting of detritylationfollowed by coupling, and sulfurization performed at ambienttemperature.

A column (diameter: 3.5 cm) was packed with a solid-support loaded with3-palmitoylamido-1-O-(4,4′-dimethoxytrityl)-2-O-succinyl propanediol(3.5 mmol based on a capacity of 400 μmol/g) that was coupled with thenucleotide monomer B′₁. Detritylation was achieved using 3%dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4column volumes in each detritylation step) and the solid-support boundnucleotide was washed with acetonitrile (amount: 5 column volumes).Coupling with the next nucleotide monomer of formula (B′_(n)) wasachieved by pumping a solution of 0.5 M 5-(ethylthio)-1H-tetrazole inacetonitrile and 0.15 M of the next nucleotide monomer of formula(B′_(n)) in the sequence, dissolved in acetonitrile, through the column.The column was washed with acetonitrile (amount: 2 column volumes). Thensulfurization was performed by pumping a solution of 0.2 M phenyl acetyldisulfide (PADS) in a 1:1 mixture of acetonitrile and 2,6-lutidinemixture through the column followed by washing the column withacetonitrile (amount: 5 column volumes).

The sulfurization was followed by a capping step. Each capping in agiven cycle used 37-47 equivalents (eq.) of the capping agent NMI, and9-11 equivalents of the capping agent B isobutryic anhydride (IBA), and1.4-1.8 equivalents of 2,6 lutidine. Capping agents A and B were pumpedthrough the column with separate pumps at different ratios such as50:50, 35:65, 65:35.

The synthesis cycle of detritylation, coupling with the next nucleotidemonomer of formula (B′_(n)) and sulfurization, and capping step wasrepeated 12 times, followed by detritylation using 3% dichloroaceticacid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes).

Upon completion of the synthesis cycle, the crude oligonucleotide on thesolid-support support was treated with a diethylamine (DEA) solutionfollowed by treatment with ammonium hydroxide solution: ethanol (3:1volume ratio) at a temperature of 55° C. The reaction mixture was agedfor 4 to 24 hours at 55° C., cooled to room temperature, and slurry wasfiltered to remove the polymeric support. The solution comprisingimetelstat in its ammonium form was subjected to the HPLC analysisprocedure of Experiment 3.

Experiment 3: Comparison of No-Capping Vs. Capping

Imetelstat obtained in Experiment 1 and Experiment 2 was analysed byHPLC. The amount of the desired full length oligonucleotide having 13nucleotides was determined and listed in the Table below for Experiment1 and Experiment 2. Also, the total amount of shortmer, specifically the12mer, was determined and listed in the Table below for Experiment 1 andExperiment 2.

HPLC analysis method:

column type: Kromasil C18, 3.5 μm particle size, 4.6×150 mm eluent:

A: 14.4 mM TEA/386 mM HFIP (hexafluoroisopropanol)/100 ppm (w/v) Na₂EDTAin water

B: 50% MeOH, 50% EtOH containing 5% IPA

Gradient:

Step Run time (minutes) % B 1 0 10 2 5 10 3 12 26 (linear) 4 35 45(linear) 5 40 50 (linear) 6 42 50 7 44 10 (linear) 8 50 10

TABLE capping vs. no-capping experiments (Experiment 1 was run twice andresults are listed as Experiment 1a and 1b). Experiment capping or MainShortmer # no capping peak % (12mer) 1a no capping 71.6% 5.5% 1b nocapping 71.2% 5.7% 2 capping 71.3% 5.6%

The HPLC analysis of Experiment 1 and Experiment 2 demonstrates thatyield and purity are comparable for the no-capping experiment vs. thecapping experiment.

Main peak % includes Full length oligonucleotide+POimpurities+depurinated impurities.

PO impurities are impurities including one or more oxophosphoramidateinternucleoside linkages instead of thiophosphoramidate internucleosidelinkages.

Solvent Use and Reaction Time

0.45 L of acetonitrile/mmol is used to prepare capping agent A andcapping agent B reagents which corresponds to approximately 25% of theoverall acetonitrile use during the preparation of the reagents. Sinceeach chemical reaction step is followed by a solvent wash, after eachcapping step too, a solvent wash takes place which is equivalent toabout 40 column volumes of the solvent. Considering that about 212column volumes of the solvent wash is done for a given synthesis run,about 19% of the wash solvent is used for the capping steps. Eachcapping step takes between 3-6 minutes. This corresponds to about 8% ofthe overall synthesis time including the 13 cycles and DEA treatment.

Experiment 4 (Detritylation Temperature)

The detritylation temperature has an impact in terms of controlling n−1and depurinated impurities. The temperature of the deblocking solutionat the entrance of the synthesizer was chosen between 17.5 and 27° C.(at 3.5 mmol scale) and the selected temperature was kept the same forall detritylation steps. The acetonitrile washing was also kept at thesame temperature of the deblocking solution. The % depurinatedimpurities increased linearly with temperature while n−1 was higher atlower temperatures.

Temperature n-1% Depurinated Impurity % 17.5 10.7 5.3 19 7.6 6.4 22 5.48.7 25 6.1 10.8 27 5.3 12.3

Experiment 5 (Sulfurization Step Temperature)

In the experiments below, the temperature (RT means room temperature) ofthe PADS solution used in the sulfurization reactions was tested for the% of less favourable PO impurities (these are impurities where phosphoroxidation occurred instead of sulfurization). Lower temperature resultsin lower PO %.

Sulfurization temperature (° C.) PO % RT 7.2 RT 8.1 RT 6.9 RT 8.8 19 6.519 6.3

imetelstat and imetelstat sodium SEQ ID NO: 1 5′-R-TAGGGTTAGACAA-NH₂-3′

wherein R represents palmitoyl [(CH₂)₁₄CH₃] amide is conjugated throughan aminoglycerol linker to the 5′-thiophosphate group of an N3′→P5′thiophosphoramidate (NPS)-linked oligonucleotide.

1. A method of synthesizing the N3′→P5′ thiophosphoramidateoligonucleotide imetelstat of formula imetelstat

  B₁ = T B₁₀ = A B₂ = A B₁₁ = C B₃ = G B₁₂ = A B₄ = G B₁₃ = A B₅ = G  B₆ = T T = thymine B₇ = T A = adenine B₈ = A G = guanine B₉ = G C =cytosine

the method comprises of a) providing a first 3′-amino protectednucleotide attached to a solid-phase support of formula (A) wherein PGis an acid-labile protecting group;

b) deprotecting the protected 3′-amino group to form a free 3′-aminogroup;

c) reacting the free 3′-amino group with a protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramiditemonomer of formula (B′_(n)) wherein n=2 to form an internucleosideN3′→P5′-phosphoramidite linkage;

d) sulfurization of the internucleoside phosphoramidite group using anacyl disulfide to form a N3′→P5′ thiophosphoramidate; e) repeating 11times in successive order the deprotection step b), the coupling step c)with a protected3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramiditemonomer of formula (B′_(n)) wherein the protected nucleoside base B′ inmonomer (B′_(n)) is successively the protected nucleobase B₃ to B 13 inthe respective 11 coupling steps, and the sulfurization step d); f)removing the acid-labile protecting group PG; and g) cleaving anddeprotecting imetelstat from the solid-phase support; characterized inthat no additional capping step is performed in any of the reactionsteps a) to e). 2-13. (canceled)